CN104957188B - 植物病原体抑制剂组合和使用方法 - Google Patents
植物病原体抑制剂组合和使用方法 Download PDFInfo
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- CN104957188B CN104957188B CN201510276279.0A CN201510276279A CN104957188B CN 104957188 B CN104957188 B CN 104957188B CN 201510276279 A CN201510276279 A CN 201510276279A CN 104957188 B CN104957188 B CN 104957188B
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Abstract
本发明涉及植物病原体抑制剂组合和使用方法。使用含有诱导植物对植物病原体的抗性的蒽醌衍生物的植物提取物和抗微生物剂、生物控制剂和/或具有杀真菌活性的表面活性剂来调控植物病原体感染的组合、组合物和使用方法。
Description
本申请是申请日为2010年7月29日，申请号为201080043530.4，发明名称为“植物病原体抑制剂组合和使用方法”的申请的分案申请。
技术领域
本文公开了使用含有诱导植物对植物病原体的抗性的蒽醌衍生物的植物提取物和抗微生物剂、生物控制剂和/或具有杀真菌活性的表面活性剂来调控植物病原体感染的组合、组合物和使用方法。
背景技术
植物对植物病原体的抗性
植物已进化了对由感染因子诸如细菌、真菌和病毒引起的疾病的抗性的高度有效机制。这一抗性可由几种机制引起，其中最著名的是***获得抗性(SAR；Ross，1961；Durrant和Dong，2004)和诱导***抗性(ISR；van Loon等人，1998)。在最简单的情形中，诱导物是植物病原体本身，在其他情形中，诱导物可以是化学化合物(水杨酸，也称为BTH的苯并(1，2，3)噻二唑-7-硫代羧酸S-甲酯(benzo(1，2，3)thiadiazole-7-carbothioic acidS-methyl ester))或物理影响诸如水或热胁迫(Walters等人，2005)。显示诱导***抗性依赖于低水平代谢扰动(metabolic perturbation)的逐渐表达和持续。与在施加位点引发并可导致局部保护的植物抗毒素积累的引发物不同，***抗性的诱导物使植物整体敏感以在感染后快速响应。这些响应包括植物抗毒素积累、木质化和增强的几丁质酶和葡聚糖酶活性。
由Marrone Bio Innovations，Inc.)以MILS和销售的虎杖(giant knotweed，Reynoutria sachalinensis)提取物提供对葫芦科植物(cucurbit)和其他作物的白粉菌和其他植物疾病的控制，主要是通过诱导植物中真菌毒性苯酚化合物的积累(Daayf等人，1995；Wurms等人1999；Schmitt，2002)。
最近，配制的虎杖提取物还已显示在不同作物和植物病原体包括小麦白粉菌中诱导抗性的极大效力(Vechet等人，2009)。除了ISR作用模式之外，最近还已显示配制的虎杖提取物具有对小麦白粉菌(Blumeria graminis f.sp.tritici；Randoux等人，2008)的直接抑真菌作用。
杀真菌剂抗性
杀真菌剂抗性是害虫、包括植物病原体中的常见现象。当频繁使用杀真菌剂、尤其是具有单位点作用模式的那些时，靶病原体可由于高选择压而适应于该杀真菌剂。预计害虫可在5-50代内发展出对杀虫剂的抗性(May，1985)。大多数植物病原体在一个生长季内符合这一范围并因此可快速发展出杀真菌剂抗性。例如，苯菌灵在其首次注册用于商业使用后仅花了一年就丧失对控制葫芦科植物白粉菌的效力(McGrath，2001)。
自从1996年引入以来，醌外部抑制剂(Quinone outside inhibitor，也称为QoI杀真菌剂或嗜球果伞素(strobilurin))已广泛用于控制农业上重要的真菌病原体。嗜球果伞素通过以下阻断呼吸途径：抑制线粒体中的细胞色素bc1复合体，从而阻断呼吸链中的电子传递过程和由于缺乏腺苷三磷酸(ATP)而引起能量不足(Bartlett等人，2002)。嗜球果伞素和其他具有单位点作用模式的杀真菌剂诸如脱甲基化抑制剂(DMI)易于在植物病原体中发展抗性。迄今为止，几种植物病原性真菌已对嗜球果伞素(Tuttle McGrath，2003；Fraaije等人，2003)和DMI杀真菌剂(Schnabel等人，2004)发展出田间抗性，并且世界范围内已进行了相当的努力来发展关于如何有计划和轮流地(in programs and rotations)组合杀真菌剂和其他抗真菌化合物以使抗性发展的风险最小的详细建议的合适的抗性管理策略(Tuttle McGrath，2006；Wyenandt等人，2009)。
控制杀真菌剂抗性的方法
管理杀真菌剂抗性的最常见策略是组合(预混合物或桶混合物(tank mix))使用易于发展抗性的位点特异性杀真菌剂。除了抗性管理以外，桶混合物还提供一种杀真菌剂失败的情形下的补偿机制以及降低剂量以降低对病原体的选择压的方式(van den Bosch和Gilligan，2008)。在一些情形中，桶混合物中或轮流的单和多位点杀真菌剂的组合可提供加成的或甚至协同的相互作用(Gisi，1996)。Holb和Schnabel(2008)在使用DMI杀真菌剂和元素硫的桶混合物的田间研究中能够显示改善的褐腐病(桃褐腐病菌(Moniliniafructicola))的控制，而Reuveni(2001)展示了将嗜球果伞素和多氧菌素B杀真菌剂与硫组合使用控制油桃白粉菌的益处。
已在与其他SAR/ISR产物以及与生物控制剂(BCA)的桶混合物中和轮流地测试了植物防御反应诱导物诸如虎杖提取物(Hafez等人，1999；Belanger和Benyagoub，1997；Schmitt等人，2002；Schmitt和Seddon，2005；Bardin等人，2008)。这些研究的目的主要是展示不同类型的植物提取物与生物控制剂的相容性。Konstatinidou-Doltsinis等人(2007)针对葡萄上的白粉菌测试了与Pseudozyma flocculosa产物轮流的虎杖产物，并发现两种产物的交替施加改善了虎杖的效力。在同一研究中，硫和虎杖轮流的交替不具有有益效应。Belanger和Benyagoub(1997)发现，当针对温室中的黄瓜白粉菌使用时，酵母样真菌Pseudozyma flocculosa与虎杖相容。相似地，Bokshi等人(2008)针对黄瓜白粉菌评估了获得性***抗性激活物苯并噻二唑和的组合效应，并发现与苯并噻二唑轮流使用的在田间提供对白粉菌的有效控制措施。然而，基于采集的疾病严重度和产量数据，无法确定该正效应是加成性的或协同性的。
杀虫剂协同作用已被定义为“其中生物体对杀虫剂组合的总应答大于个体组分之和的两种或多种化合物的同时作用”(Nash，1981)。因此，当杀真菌剂协同地相互作用时，在低于每种个体杀真菌剂标签比例(label rate)下实现高水平疾病控制。通常，最佳效应在具有不同作用模式(MOA)的杀真菌剂组合中实现，但是协同性也在组合使用具有相似作用模式的产物时展示(De Waard，1996)。杀真菌剂协同作用主要是在实验室研究中展示的(Samoucha和Cohen，1984；Gisi，1996)，但是在一些情形下(Karaogladinis和Karadimos，2006；Burpee和Latin，2008)，也在田间研究中发现了协同作用。另外，除杀真菌剂外的抗真菌化合物(碳酸氢盐和精炼石油镏出物(refined petroleum distillate))也展示抵抗玫瑰白粉菌和黑斑病(Horst等人，1992)。
发明内容
公开并要求保护包含以下的组合：(a)源自植物的提取物，其中所述提取物含有诱导植物对植物病原体(phytopathogen)的抗性(也称为“植物病原体(plant pathogen)”)的一种或多种蒽醌衍生物，和(b)选自由以下组成的组的一种或多种抗植物病原体剂：(i)非-benzodiathiazole、非-维生素E、非-有机磷抗微生物剂，其缺乏或换言之不含非-元素、非-可湿性的硫，(ii)具有杀真菌活性的表面活性剂，和(iii)非-芽孢杆菌属(Bacillus)、非-假单胞菌属(Pseudomonas)、非-短芽孢杆菌属(Brevabacillus)、非-轮枝菌属(Lecanicillium)、非-白粉寄生孢属(Ampelomyces)、非-茎点霉属(Phoma)、非-Pseudozyma生物控制剂(如，源自链霉菌属(Streptomyces sp.)、伯克氏菌属(Burkholderia sp.)、木霉属(Trichoderma sp.)、粘帚霉属(Gliocladium sp.)的剂或具有杀真菌和/或杀昆虫活性天然油或油产物)。
在具体实施方案中，组合包含：(a)源自蓼科(Polygonaceae)的提取物和(b)非-benzodiathiazole、非-维生素E、非-有机磷抗真菌和/或抗微生物剂，其缺乏或不含非-元素或非-可湿性的硫。
在一个具体实施方案中，组合包含：(a)源自蓼科的提取物(如虎杖)，和(b)单位点杀真菌剂和/或多位点杀真菌剂，其可包括但不限于腈菌唑、喹氧灵、嘧菌酯、阿拉酸式苯-S-甲基、精甲霜灵(mefenoxam)、氟菌唑、咯菌腈(fludioxonil)、丙环唑。
在另一具体实施方案中，组合包含：(a)源自蓼科的提取物(如虎杖)和(b)具有杀真菌和/或杀昆虫活性天然油或油产物。
在又一特定实施方案中，组合是组合物，特别是用于调控植物病原体或真菌感染的组合物。本发明还涉及提取物和抗植物病原体剂在配制这些组合物中的用途。
本发明另外涉及用于调控植物病原体感染的协同组合，该协同组合包含：(a)源自植物的提取物，其中所述植物含有诱导植物对植物病原体的抗性的蒽醌衍生物，和(b)非-维生素E、非-有机磷抗微生物剂(如，抗真菌和/或抗微生物剂)，其缺乏或不含非-元素或非-可湿性的硫。在特定实施方案中，抗微生物剂是benzodiathiazole(如，阿拉酸式苯-S-甲基)、***(如，丙环唑)或嗜球果伞素(如，嘧菌酯)。
以上提及的组合还可配制成组合物。
本发明还涉及用于在植物中调控植物病原体感染的方法，包含向植物和/或其种子和/或用于培养所述植物的基质施加有效调控所述植物病原体感染的量的如上所述的本发明的组合。
在特定实施方案中，本发明涉及用于在植物中调控真菌和/或细菌感染的方法，包含向植物和/或其种子和/或用于培养所述植物的基质施加有效调控所述真菌和/或细菌感染的本发明的组合。
提取物和所述抗植物病原体剂(如，抗真菌和/或抗微生物剂)可以顺序地、同时地或间歇组合地施用。如本文所定义，“植物病原体感染”意指植物被植物病原性细菌、真菌、昆虫、线虫和/或软体动物的感染。
本发明还涉及降低植物病原体(如，真菌和/或细菌)对以下的抗性的方法：(i)非-维生素E、非-有机磷抗微生物剂，其缺乏或换言之不含非-元素或非-可湿性的硫，(ii)具有杀真菌活性的表面活性剂，和/或(iii)非-芽孢杆菌属、非-假单胞菌属、非-短芽孢杆菌属、非-轮枝菌属、非-白粉寄生孢属、非-茎点霉属、非-Pseudozyma生物控制剂(如，源自链霉菌属、伯克氏菌属、木霉属、粘帚霉属的剂或具有杀真菌和/或杀昆虫活性的天然油或基于油的产物)，该方法包含：向有相应需要的植物施用有效降低所述抗性发生的量的本发明的组合。在特定实施方案中，本发明涉及用于降低真菌和/或细菌对非-元素或非-可湿性的硫、非-benzodiathiazole、非-维生素E、非-有机磷抗真菌和/或抗微生物剂的抗性的方法。在又一特定实施方案中，本发明涉及使用本发明的组合降低真菌和/或细菌对具有杀真菌和/或杀昆虫活性的天然油或基于油的产物的抗性的方法。这可通过降低发生频率或比率实现。植物病原体对以上提及的抗病原体剂的抗性可被降低至少50％。
本发明还涉及(a)源自植物的提取物，其中所述提取物含有诱导植物对植物病原体的抗性的蒽醌衍生物，和(b)选自由以下组成的组的一种或多种抗植物病原体剂：(i)非-benzodiathiazole、非-维生素E、非-有机磷抗微生物剂，其缺乏非元素、非-可湿性的硫，(ii)具有杀真菌活性的表面活性剂，和(iv)非-芽孢杆菌属、非-假单胞菌属、非-短芽孢杆菌属、非-轮枝菌属、非-白粉寄生孢属、非-茎点霉属、非-Pseudozyma生物控制剂，用于制备调控植物病原体感染的组合物的用途，以及(a)源自植物的提取物，其中所述植物含有诱导植物对植物病原体的抗性的蒽醌衍生物和(b)benzodiathiazole抗微生物剂用于制备调控植物病原体感染的协同组合物的用途。
本发明还涉及包含以下的试剂盒：(a)源自蓼科的提取物，和(b)选自由以下组成的组的一种或多种抗植物病原体剂：(i)非-benzodiathiazole、非-维生素E、非-有机磷抗微生物剂，其缺乏非元素或非-可湿性的硫，(ii)具有杀真菌活性的表面活性剂，和(iii)非-芽孢杆菌属、非-假单胞菌属、非-短芽孢杆菌属、非-轮枝菌属、非-白粉寄生孢属、非-茎点霉属、非-Pseudozyma生物控制剂。该试剂盒还可包含包装说明。
具体实施方式
当提供值的范围时，应理解，除非上下文另外说明，该范围上限和下限之间至下限单位的十分之一的每个间插值和该所称范围的任何其他所称值或间插值涵盖在本发明内。可独立地包括在较小范围中的这些更小范围的上限和下限也涵盖在本发明内，并经受所称范围内的任何具体排除的限值。当所称范围包括所述限值的一个或两个时，排除所包括的限值的任一个两个的范围也包括在本发明中。
除非另外定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常所理解的含义相同的含义。尽管在本发明的实践或检验中也可使用与本文所述的那些方法和材料相似或等同的任何方法和材料，本文描述了优选的方法和材料。另外，尽管参考具体实施方案描述了本发明，但是其细节不应解读为限制，因为明显技术人员可以使用不同的等同物、变化和修饰而仍在本发明范围内。
贯穿本说明书引用了不同的参考文献，其中每一篇参考文献通过引用整体并入本文。
必须注意，如本文和所附权利要求书中所用，除非上下文另外清楚指明，否则单数形式“一种(a)”、“一个(and)”和“该(the)”包括复数指代。例如，“一种真菌(a fungus)”还涵盖“多种真菌(fungi)”。
如本文所定义，术语“调控”用于意指改变植物病原体感染的量或植物病原体感染的传播速率。
提取物
本发明的组合、组合物和方法中使用的植物提取物含有蒽醌衍生物作为用于抵抗植物害虫，特别是植物植物病原体诸如植物病原性细菌、真菌、昆虫、线虫的生物化学农业产品和/或作为杀软体动物剂。“含有”还涵盖产生所述蒽醌衍生物的提取物。在特定实施方案中，本发明的组合物和方法中使用的本发明的蒽醌衍生物是主要活性成分或主要活性成分之一。
蒽醌衍生物包括但不限于大黄素甲醚、大黄素、大黄酚、ventiloquinone、大黄素糖苷、大黄酚糖苷、大黄素甲醚糖苷、3，4-二羟基-1-甲氧基蒽醌-2-甲醛、虎刺醛。这些衍生物共有以下的相似结构：
其中R1、R2、R3、R4、R5、R6、R7和R8是氢、羟基、羟基烷基、卤素、羧基、烷基、烷氧基、烯基、烯氧基、炔基、炔氧基、杂环基、芳香族或芳族基、糖诸如葡萄糖，
在特定实施方案中，本发明涉及包含在源自包括但不限于以下植物科的提取物中的蒽醌衍生物：蓼科、鼠李科(Rhamnaceae)、豆科(Fabaceae)、独尾草科(Asphodelaceae)和茜草科(Rubiaceae)等等。这些化合物可来自植物的任何部位诸如叶、茎、树皮、根和果实。植物材料可以是湿的和干的，但优选是干的植物材料。为了符合生物化学农业产品要求，提取和纯化中使用的溶剂和工艺必须符合美国国家有机计划(National Organic Program，NOP)[http：//www.ams.usda.gov/AMSv1.0/nop，]的要求。
在更具体的实施方案中，植物提取物源自蓼科的成员。如本文所定义、“源自”意指直接分离自或获自特定的来源，或可选地具有分离自或获自特定来源的物质或生物体的鉴定特征。在特定实施方案中，所述组合中的提取物含有蒽醌衍生物大黄素甲醚和任选地大黄素。蓼科的成员包括但不限于：Acetosella、珊瑚藤属(Antigonon)、木蓼属(Aristocapsa)、Bilderdykia、黄珊瑚藤属(Brunnichia)、Centrostegia、Chorizanthe、海葡萄属(Coccoloba)、Coccolobis、Coccolobo、Corculum、Dedeckera、Delopyrum、Dentoceras、Dodecahema、刺酸模属(Emex)、Eriogonum、Fafopyrum、荞麦属(Fagopyrum)、首乌属(Fallopia)、Gilmania、Goodmania、Harfordia、Hollisteria、冰岛蓼属(Koenigia)、Lastarriaea、Mucronea、竹节蓼属(Muehlenbeckia)、Nemacaulis、山蓼属(Oxyria)、Oxytheca、Perscarioa、春蓼属(Persicaria)、Pleuropterus、Podopterus、Polygonella、萹蓄属(Polygonum)、Pterostegia、大黄属(Rheum)、酸模属(Rumex)、Ruprechtia、Stenogonum、Systenotheca、Thysanella、Tovara、Tracaulon、蓼树属(Triplaris)和甚至更优选的实施方案，提取物可源自虎杖属(Reynoutria，也称为Fallopia)或大黄属物种。在最具体的实施方案中，提取物源自虎杖。
抗植物病原体剂
配制的提取物(诸如以商标名和销售的产品)然后可与具有单位点、多位点或未知作用模式的其他抗植物病原体剂组合使用：植物提取物、生物杀虫剂、无机作物保护剂(诸如铜)、表面活性剂(诸如鼠李糖脂；Gandhi等人，2007)或拥有杀虫剂性质的天然油诸如石蜡基油和茶树油或化学杀真菌剂或杀细菌剂。如本文所定义，“抗植物病原体剂”是调控植物上的植物病原体生长或可选地防止植物被植物病原体感染的剂。植物病原体包括但不限于真菌、细菌、病毒、昆虫、线虫和/或软体动物。
在特定实施方案中，抗植物病原体剂是可选地称为生物控制剂的生物杀虫剂。该生物控制剂在更具体的实施方案中是非-芽孢杆菌属、非-假单胞菌属、非-短芽孢杆菌属、非-轮枝菌属、非-白粉寄生孢属、非-茎点霉属、非-Pseudozyma生物控制剂，是源自链霉菌属、伯克氏菌属、木霉属、粘帚霉属的剂。可选地，该剂是具有杀真菌和/或杀昆虫活性的天然油或油产物(如，石蜡基油、茶树油、柠檬草油、丁香油、肉桂油、柠檬油、迷迭香油)。
如上所述的，抗植物病原体剂可以是单位点抗真菌剂，其可包括但不限于苯并咪唑、脱甲基化抑制剂(DMI)(如，咪唑、哌嗪、嘧啶、***)、吗啉、羟基嘧啶、苯胺并嘧啶(anilinopyrimidine)、硫代磷酸酯、醌外部抑制剂、喹啉、二羧酰亚胺(dicarboximide)、羧酰亚胺(carboximide)、苯基酰胺、苯胺并嘧啶、苯基吡咯、芳族烃、肉桂酸、羟基苯胺、抗生素、多氧菌素、酰基胺(acylamine)、酞酰亚胺、苯环型化合物(benzenoid，二甲苯基丙氨酸)。在更具体的实施方案中，抗真菌剂是选自由以下组成的组的脱甲基化抑制剂：咪唑(如，氟菌唑)、哌嗪、嘧啶和***(如双苯***醇、腈菌唑、戊菌唑、丙环唑、***酮、糠菌唑、环唑醇、烯唑醇、腈苯唑、己唑醇、戊唑醇、四氟醚唑、丙环唑)。在最具体的实施方案中，抗真菌剂是腈菌唑。在又一特定实施方案中，抗真菌剂是醌外部抑制剂(如，strobulurin)。strobulurin可包括但不限于嘧菌酯、醚菌酯或肟菌酯。在又一特定实施方案中，抗真菌剂是醌，如喹氧灵(5，7-二氯-4-喹啉基4-氟苯基醚)。
在又一另外的实施方案中，抗微生物剂是选自由以下组成的组的多位点非-无机、化学杀真菌剂：腈(如，氯腈或咯菌腈)、喹喔啉、磺酰胺、膦酸酯(phosphonate)、亚磷酸酯、二硫代氨基甲酸酯、chloralkythio、苯基吡啶-胺(phenylpyridin-amine)、氰基-乙酰胺肟(cyano-acetamide oxime)。
在又一实施方案中，抗植物病原体剂是抗细菌剂。该抗细菌剂包括但不限于氨基甲酸酯、有机磷酸酯、环状二烯有机氯化物(cyclodiene organochlorides)、苯基吡唑、拟除虫菊酯、除虫菊酯、新烟碱(neonicotinoids)、硝基胍(nitroguanadines)、烟碱、多杀菌素(Spinosyn)、糖苷、保幼激素类似物和其他昆虫生长调节物、吡啶偶氮甲碱(pyridineazomethine)、吡啶羧酰胺(pyridine carboxamide)、四嗪、噻唑烷酮(thiazolidinone)、2，4-diphenyloxzoline衍生物、有机锡、吡咯、噻嗪酮(buprofezin)、氟蚁腙(hydramethylnon)、naphtoquinon衍生物、哒嗪酮、苯氧基吡唑(phenoxypyrazole)、特窗酸(tetronic acid)、肼基甲酸酯(carbazate)、鱼藤酮、有机氯-二苯基脂族化合物(organochlorine-diphenylaliphatics)。
用途
所述植物提取物或配制的产品可与其他组分或多种组分以桶混合物或以在生长季中具有预定顺序和施加间隔的计划(称为轮流的顺序施加)同时使用。当在组合中与以上提及的杀虫剂产品一起使用时，在低于产品标签建议的浓度下，两种或更多种产品(其中之一是所述植物提取物)的组合效力在优选的实施方案中高于每种个体组分的效应加在一起。因此，杀虫剂效应被这两种(或更多种)产品之间的协同作用增强，并且在植物病原性株系中发展杀虫剂抗性的风险被降低。
本发明的范围内保护的靶植物包括例如以下植物物种：禾本植物(小麦、大麦、黑麦、燕麦、水稻、高粱和相关作物)、甜菜(食用甜菜(sugar beet)和饲用甜菜(fodderbeet))、马铃薯和软水果(苹果、梨、李子、桃、杏、樱桃、草莓、覆盆子和黑莓)、豆类植物(黄豆(bean)、小扁豆(lentil)、豌豆和大豆(soybean))、油料植物(油菜、芥菜、罂粟、橄榄、向日葵、椰子、蓖麻油植物、可可豆和花生)、葫芦科植物(黄瓜、甜瓜、南瓜、茄子)、纤维植物(棉花、亚麻、***和黄麻)、柑橘类水果(橙子、柠檬、葡萄柚和蜜桔)、蔬菜(菠菜、莴苣、芦笋、白菜、胡萝卜、洋葱、番茄、土豆和红辣椒)、樟科植物(鳄梨、肉桂和樟脑)或植物诸如迷宫植物(maze)、烟草、坚果、咖啡、甘蔗、茶、藤(vine)、蛇麻(hop)、香蕉和天然橡胶植物、以及观赏植物(菊科的)、抗土壤侵蚀或干燥和在树木和多年生植物(水果栽植、蛇麻栽植、迷宫田(maze field)、葡萄园等)栽培中有用的草坪(areas of grass)或普通的低覆盖作物(low cover crop)。
施加本发明产品的组合的优选方法是用或不用载体叶面施加(喷雾(spraying)、雾化(atomizing)、喷洒(dusting)、播散(scattering)或倾灌(pouring))。施加的次数和施加比例(rate of application)取决于被病原体感染的风险。例如，本专利中覆盖的组合和混合物的叶面杀虫剂处理可以比产品标签中建议的比例低25至10,000-倍的比例每7至14天进行一次。本发明的目标产品混合物和组合也可通过用含有活性成分的液体制剂浸渍种子或用固体制剂包衣种子来施加至种子。在特殊的情形中，施加的另外类型也是可能的。这些包括土壤浇灌(soil drench)或植物茎或芽的选择性处理。
本发明的混合物和适当时固体或液体佐剂以已知方式制备。例如，混合物可通过均匀混合和/或研磨活性成分与增量剂诸如溶剂、固体载体和适当时表面活性化合物(表面活性剂)来制备。组合物还可含有另外的成分诸如稳定剂、粘性调节剂、粘合剂、佐剂以及肥料或其他活性成分以获得特殊的效果。
实施例
如下文将阐述的，为满足前述目标和优点，通过以产生协同或加成效应的比例使用两种或多种产品来增加它们的效力的方法的实例。这里描述的组合物和方法已被证明有效降低温室种植的黄瓜(Cucumis sativus)的疾病发病率和严重度，但是这一概念可有效用于其他植物品种和物种。组合物和方法对于抵抗黄瓜白粉菌是特别有效的，但是它们也可应用于其他真菌、细菌和病毒性植物疾病，诸如灰霉病、叶斑病、细菌性萎蔫病、斑点病(scab)、炭疽病、烟草花叶病毒等。
材料和方法
白粉菌(实施例I-V和X-XI)
研究在温室中进行。协同作用研究的实验设计按照Burpee和Latin(2008)。由Sphaerotheca fuliginea引起的白粉病用于研究处理的效力。
黄瓜种子栽培品种“SMR 58”(Irwin&Sons Ag Supply，Inc.Cheshire，OR)在含有盆栽土壤混合物(potting soil mix，Rod McLellan Company，Marysville，OH)的4-英寸塑料盆中生长。在2-真叶期处理植物。不同比例的化合物用2-oz喷雾器以每株植物叶片上部2ml和下部1ml喷雾。接种前，将处理的植物在荧光灯下放置3-4小时以进行干燥。
黄瓜白粉菌病的致病病原Sphaerotheca fuliginea的分生孢子悬液通过剪下用于保存接种体的黄瓜植物的病叶来准备。将悬液调整至2.0×105个分生孢子/ml并用2-oz喷雾器以叶片上部2ml/植物施加。将接种的植物置于温室，并在25℃至30℃用4至6个重复以随机完全区组安排处理。
第一叶的疾病严重度(菌落覆盖的百分比面积)根据James(1971)评价。疾病严重度和控制百分比用方差分析(ANOVA)进行分析，并在p＝0.05水平比较处理的平均值与Fisher保护最小差异(Fisher’s Protected Least Difference，LSD)。计算协同效应并用Limpel的公式分析(Limpel等人，1962；Richer，1987)。
莴苣霜霉病(实施例VI-VII)
控制特定病原体类别卵菌(Oomycetes)的MBI-106和阿拉酸式苯-S-甲基或精甲霜灵之间的协同性用莴苣霜霉病按照Su等人(2004)测试。
为了准备霜霉病接种体，将莴苣种子以每个培养皿约20-30颗种子置于5-mm培养皿上，并用无菌水浇水，然后在萌发后供应1/2强度的Hoagland溶液。将培养皿在20℃生长室放置7-10天。将Bremia lactucae的孢子接种到莴苣子叶上，并在15℃培养7-10天以发生孢子形成。剪下具有孢子形成的子叶并置于含无菌水的falcon管中。将子叶涡旋15秒钟三次，并过滤通过100-uμm网筛以收集孢子。将溶液调整为0.5至1.0×105个孢子/ml用于接种。
四株测试植物播种于每个2-in盆中，然后在20℃放置-10天以生长子叶。当出现第一片真叶时，植物可以用于测试。
将莴苣植物用材料处理并放置至干燥或过夜。然后用孢子溶液接种植物。处理以四个重复的随机完全区组设计来安排。将接种的植物在生长室中置于黑暗中48小时，然后以12-h光周期在15℃下培养。接种八至十天后，评价子叶的疾病严重度(孢囊梗覆盖的百分比面积)。
种子处理(实施例VIII-IX)
下面描述的实验用大豆进行，但是相似的程序可用于其他作物诸如谷物、玉米、棉花和马铃薯。
实验在温室中按照Hwang等人(2006)开发的程序进行。Rhizoctonia solani的分离株在马铃薯右旋糖琼脂平板上生长并培养5天用于接种燕麦或小麦谷粒。一升谷粒在室温(25℃)下浸泡过夜并用自来水润洗三次。8×12-in高压灭菌袋用谷粒装至1/3满，在121℃下高压灭菌15min。然后将高压灭菌的谷粒每袋用R.solani的5天培养物的5个1×1cm塞(plug)接种并在室温(25℃)下培养5天。将袋开口放置在层流柜中以使谷粒完全干燥。
将10g待包衣的空白种子样品置于烧瓶中。将包衣剂1171-O(BeckerUnderwood Ltd.，Ames，IA)与目标化合物一起置于烧瓶中，剧烈振荡烧瓶使得种子包上化合物。用栽培品种“White Lion”(Kitazawa Seed Co.，Oakland，CA)或“Viking 2265”(Johnny’s Selected Seeds，Winslow，ME)的新鲜种子样品在相同烧瓶中重复程序用于处理。在包衣种子后，在种植之前，将它们晾干。
为了准备土壤，将500毫升含有接种体的干燥的谷粒掺和15秒钟三次，并将粉末与无菌沙以1∶1(v/v)混合以稀释接种体。对于大豆，沙混合物还用于以1∶350至1∶800(接种体∶土壤)的不同比例准备土壤混合物，以在重复的测试中产生不同的疾病水平。
将包衣的种子种植在具有感染土壤的盆中。每个处理三个重复，处理以完全随机区组设计安排，并在温室中置于25至30℃。
10至20天后，取决于疾病压力和温度，评价并比较每个处理的出苗(emergence)。通过称重地上部测量生物量，对于植物材料的每个重复进行测量。
实施例I.配制的虎杖提取物和腈菌唑之间的协同效应(测试I).
以比1500×和2000×的标签比例低10-倍的比例稀释的MBI-106(由Marrone BioInnovations，Inc.，Davis，CA以SC销售的配制的虎杖提取物)和0.25ug/ml、0.1ug/ml，和0.05ug/ml(比建议的标签比例低450至2,250-倍)浓度的腈菌唑(配制为40W，Dow AgroSciences LLC，Indianapolis，IN)单独施加或以桶混合物施加。
疾病严重度在与0.25ug/ml腈菌唑组合的1500×和2000×的MBI-106中比它们单独使用时显著降低(表1)。发现0.25ug/ml和0.05ug/ml比例的腈菌唑桶混合的MBI-1062000×组合的控制效力显著增加(表2).
实施例II.虎杖提取物和腈菌唑之间的协同效应(测试II).
在使用MBI-106(配制为SC)和腈菌唑(配制为40W)的第二测试中，发现当2000×(比标签比例低10-倍)稀释的MBI-106与0.25、0.1和0.05ug/ml比例的腈菌唑组合时和当1500×的MBI-106与0.05ug/ml的腈菌唑桶混合时的更高效力(表3)。当以1500×和2000×稀释的MBI-106与0.05ug/ml的最低比例的腈菌唑组合时展示协同效应(表4)。
实施例III.配制的虎杖提取物和喹氧灵之间的协同效应
以1500×或2000×稀释的MBI-106单独施加或与0.05和0.01ug/ml(比标签比例低2,000至10,000-倍)的喹氧灵(配制为Dow AgroSciences LLC，Indianapolis，IN)单独或以桶混合物组合施加。结果显示于5和6。最显著的协同效应和增强的疾病控制发现于2000×的虎杖产物稀释与0.01ug/mL的喹氧灵。喹氧灵具有影响早期细胞信号传导中的G-蛋白的新颖多位点作用模式。
实施例IV.配制的虎杖提取物和嘧菌酯之间的协同效应
以1500×或2000×稀释的MBI-106单独施加，或与0.25、0.5、1.0、5.0和10ug/mL(比建议的标签比例低25至1020-倍)的嘧菌酯(配制为SyngentaCorporation，Wilmington，DE)单独或以桶混合物组合施加。结果显示于7和8。在所有测试的组合中，2000×稀释的虎杖提取物和5.0或0.5ug/mL的嘧菌酯的混合物提供最大的协同性，并且与单一化合物使用数据相比，杀真菌效力显著高于预期。
实施例V.配制的虎杖提取物和氟菌唑之间的协同效应。
2500×稀释(比标签比例低10-倍)的MBI-106(由Marrone Bio Innovations，Inc.，Davis，CA以SC销售的配制的虎杖提取物)和1.0ug/ml、0.5ug/ml和0.25ug/ml(比建议的标签比例低150至600-倍)浓度的氟菌唑(配制为480SC，Chemtura Corporation，Middlebury，CT)单独或以桶混合物施加。
疾病严重度在2500×稀释的MBI-106与氟菌唑组合喷雾的处理中与单独使用MBI-106的处理中相比显著降低(P＜0.0001)(表9)。发现与1.0ug/ml、0.5ug/ml和0.25ug/ml比例的氟菌唑桶混合的MBI-1062500×组合的效力协同效应(表10)。
实施例VI.在控制莴苣霜霉病中虎杖提取物和阿拉酸式苯-S-甲基之间的协同效应。
MBI-106(由Marrone Bio Innovations，Inc.，Davis，CA以SC销售的配制的虎杖提取物)以200×稀释单独使用或与25ug/ml浓度的阿拉酸式苯-S-甲基(配制为Syngenta Crop Protection，Inc.，Greensboro，NC)组合使用以控制莴苣霜霉病。
疾病严重度在200×稀释的MBI-106与阿拉酸式苯-S-甲基的组合中与它们单独使用的处理中相比显著降低(p＝0.0004)(表11)。MBI-106200×和阿拉酸式苯-S-甲基的桶混合物存在效力的协同效应(表12)。
实施例VII.在控制莴苣霜霉病中虎杖提取物和精甲霜灵之间的协同效应。
为了研究虎杖属和MBI-106的协同效应，Marrone Bio Innovations，Inc.，Davis，CA的配制的产品ME以400×稀释比例与37.5μg/ml、75.0μg/ml和150μg/ml浓度的精甲霜灵(配制为RIDOMILSyngenta Crop Protection，Inc.，Greensboro，NC)组合使用以控制莴苣霜霉病。
疾病严重度在400×稀释的MBI-106与精甲霜灵的组合中与它们单独在每种浓度使用的处理相比显著降低(p＜0.0001)(表13)。发现MBI-106和每种浓度的精甲霜灵的组合存在MBI-106200×和精甲霜灵桶混合物的效力协同效应(表14)。
实施例VIII.虎杖提取物作为种子处理剂和其在控制Rhizoctonia solani中与嘧菌酯的协同性。
虎杖用乙醇5％(w/w)提取，并单独或与0.0298g/kg种子的嘧菌酯(Syngenta Crop Protection，Inc.，Greensboro，NC)组合以0.2117g/kg种子用于种子包衣以控制大豆的Rhizoctonia solani。
出苗率在用MBI-106处理的接种种子中与接种的未处理对照相比更高，并且当与嘧菌酯组合使用时，出苗率比单独使用任一种产物时高(表15)。发现当组合使用两种材料时的协同效应(表16)。
实施例IX.虎杖提取物作为种子处理剂和与咯菌腈在控制Rhizoctonia solani中的协同性。
虎杖的乙醇提取物也以0.03175g/kg种子和0.635g/kg种子比例单独或与咯菌腈(由Syngenta Crop Protection，Inc.，Greensboro，NC配制为)组合用于种子包衣以控制大豆上的Rhizoctonia solani。出苗率和生物量在MBI-106处理的种子中与接种的未处理对照相比更高，并且当与0.0596g/kg种子比例的咯菌腈组合使用时也更高(表17)。发现当与咯菌腈组合使用时两种比例的MBI-106的协同效应(表18)。
实施例X.在控制黄瓜白粉菌中配制的虎杖提取物和丙环唑之间的协同效应。
2500×稀释的MBI-106(由Marrone Bio Innovations，Inc.，Davis，CA以ME销售的配制的虎杖提取物)和1.0ug/ml浓度的丙环唑(配制为EC，Dow AgroSciences LLC，Indianapolis，IN)单独或以桶混合物施加。
疾病严重度在2500×稀释的MBI-106与丙环唑组合喷雾的处理中显著降低(P＜0.0001)(表19)。与丙环唑桶混合的MBI-1062500×组合的控制效力存在协同效应(表20)。
实施例XI.在控制黄瓜白粉菌中虎杖提取物和quinoxifen之间的协同效应(测试II)。
MBI-106(由Marrone Bio Innovations，Inc.，Davis，CA以SC销售的配制的虎杖提取物)以2000×稀释单独使用或与三种浓度0.5、0.25和0.1ug/ml的嘧菌酯组合使用以控制黄瓜白粉菌。
2000×稀释的MBI-106与嘧菌酯组合处理中的控制效力比它们单独使用时高(表21)。MBI-106 2000×桶混合物的控制效力的协同效应存在于MBI-106和每种浓度的嘧菌酯组合(表22)。
表格
表1.MBI-106和腈菌唑(40W)单独使用或以桶混合物使用时的疾病严重度和控制百分比.1
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1第3列中的数据(严重度(％))是六个重复的平均值。一列中带有相同字母的平均值是根据Fisher保护最小显著性差异(LSD)在P＝0.05水平差异不显著的。
表2.每种产物组合的预期效力(Ee)2以及MBI-106(SC)和腈菌唑(40W)之间所检测的协同作用的统计学显著性
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2Ee是预期效力，并根据Limpel的公式Ee＝X+Y-(XY)/100(Limpel等人，1962；Richer，1987)确定。
3数据是六个重复的平均值。一列中带有相同字母的平均值是根据Fisher保护最小显著性差异(LSD)在P＝0.05水平差异不显著的。
4n.s.：不显著的
5*和***：分别在P＜0.05和0.001下显著。
表3.重复测试中MBI-106(SC)和腈菌唑(40W)单独使用或以桶混合物使用时的疾病严重度和控制百分比
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6数据是四个重复的平均值。一列中带有相同字母的平均值是根据Fisher保护最小显著性差异(LSD)在P＝0.05水平差异不显著的。
表4.重复的测试中MBI-106(SC)和腈菌唑(40W)之间的协同效应。
zEe是预期效力，并根据Limpel的公式Ee＝X+Y-(XY)/100(Limpel等人，1962；Richer，1987)确定。
**和***：分别在P＜0.01和0.001下显著的。
n.s.：不显著的
表5.MBI-106(配制的虎杖提取物)和喹氧灵()单独使用或以桶混合物使用时的疾病严重度和控制百分比。
数据是四个重复的平均值。一列中带有相同字母的平均值是根据Fisher保护最小显著性差异(LSD)在P＝0.05水平差异不显著的。
表6.MBI-106(虎杖属提取物、SC)和喹氧灵()之间的协同效应
ZEe是预期效力，并根据Limpel的公式Ee＝X+Y-(XY)/100(Limpel等人，1962；Richer，1987)确定
*和**：分别在P＜0.05和0.01下是显著的。
n.s.：不显著的。
表7.疾病严重度和使用MBI-106(虎杖提取物，SC)和嘧菌酯()时白粉菌的控制百分比。
表8.MBI-106和嘧菌酯之间的协同效应。仅包括了具有统计学显著的协同效应的处理。
表9.虎杖和氟菌唑(480SC)单独使用或以桶混合物使用时的疾病严重度和控制百分比。
第3列中的数据(严重度(％))是四个重复的平均值。一列中带有相同字母的平均值是根据Fisher保护最小显著性差异(LSD)在P＝0.05水平差异不显著的。
表10.MBI-106(SC)和氟菌唑(480SC)之间的每种产物组合的预期效力(Ee)、控制效力(E)和协同效应(E/Ee＞1.0)。
Ee是预期效力，并根据Limpel的公式Ee＝X+Y-(XY)/100(Limpel等人，1962；Richer，1987)确定。
表11.MBI-106(SC)和阿拉酸式苯-S-甲基()单独使用或以桶混合物使用时的疾病严重度和控制百分比。
表12.MBI-106(SC)和阿拉酸式苯-S-甲基()之间每种产物组合的预期效力(Ee)、控制效力(E)和协同效应(E/Ee＞1.0)。
| 桶混合物 | 控制(％) | Ee | E/Ee |
| 虎杖属提取物+阿拉酸式苯-S-甲基 | 84.2 | 51.5 | 1.6 |
表13.MBI-106(ME)和精甲霜灵(RIDOMILSL)的疾病严重度和控制百分比。
表14.MBI-106(ME)和精甲霜灵(RIDOMILSL)之间每种产物组合的预期效力(Ee)、实际控制效力(E)和检测的协同作用(E/Ee＞1.0)。
表15.大豆幼苗的出苗以及MBI-106(乙醇提取物)和嘧菌酯()对猝倒病的控制。
表16.MBI-106(乙醇提取物)和嘧菌酯()之间的每种产物组合的预期效力(Ee)、实际控制效力(E)和检测的协同作用(E/Ee＞1.0)。
表17.大豆幼苗的出苗以及MBI-106(虎杖属乙醇提取物)和咯菌腈()对猝倒病的控制。
表18.MBI-106(乙醇提取物)和咯菌腈()之间的每种产物组合的预期效力(Ee)、实际控制效力(E)和检测的协同作用(E/Ee＞1.0)。
表19.控制黄瓜白粉菌中虎杖和丙环唑()单独使用或以桶混合物使用时的疾病严重度和控制百分比。
表20.MBI-106(ME)和丙环唑()之间的每种产物组合的预期效力(Ee)、控制效力(E)和协同效应(E/Ee＞1.0)。
表21.控制黄瓜白粉菌中(虎杖和嘧菌酯()单独使用或以桶混合物使用时的疾病严重度和控制百分比
表22.MBI-106(SC)和嘧菌酯()(测试II)之间的每种产物组合的预期效力(Ee)、控制效力(E)和协同效应(E/Ee＞1.0)。
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Claims (6)
1.一种组合物，包含：(a)1500或2000倍稀释的以SC销售的大虎杖(Reynoutria sachalinensis)植物提取物MBI-106，所述大虎杖植物提取物MBI-106含有诱导植物对真菌感染的抗性的大黄素甲醚，和(b)0.25μg/ml、0.1μg/ml或0.05μg/ml配制为40W的腈菌唑，并且其中所述组合物提供经由确定E/Ee而测量的协同效应，且E/Ee大于1.0。
2.根据权利要求1所述的组合物，其中所述组合物包含(a)2000倍稀释的大虎杖(Reynoutria sachalinensis)植物提取物MBI-106和(b)0.05μg/ml腈菌唑。
3.根据权利要求1所述的组合物，其中组分(a)和组分(b)以桶混合物施加。
4.一种用于在植物中调控真菌感染的方法，包含向植物和/或其种子和/或用于培养所述植物的基质施加有效调控所述真菌感染的量的：(a)1500或2000倍稀释的以SC销售的大虎杖(Reynoutria sachalinensis)植物提取物MBI-106，所述大虎杖植物提取物MBI-106含有诱导植物对真菌感染的抗性的大黄素甲醚，和(b)0.25μg/ml、0.1μg/ml或0.05μg/ml配制为40W的腈菌唑，并且其中所述组分(a)和组分(b)提供经由确定E/Ee而测量的协同效应，且E/Ee大于1.0。
5.根据权利要求4所述的方法，包含施加(a)2000倍稀释的大虎杖(Reynoutriasachalinensis)植物提取物MBI-106和(b)0.05μg/ml腈菌唑。
6.根据权利要求4所述的方法，其中组分(a)和组分(b)以桶混合物施加。
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| CN115088724B (zh) * | 2022-08-26 | 2022-11-15 | 山东康惠植物保护有限公司 | 一种用于黄瓜白粉病的含四氟醚唑和大黄酚的杀菌剂 |
| CN115918665B (zh) * | 2023-01-05 | 2024-04-30 | 河南科技学院 | 一种防治小麦茎基腐病的小麦拌种剂 |
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